1. Volume 2, Issue 2, Pages 223-232 (August 1998)
Xeroderma Pigmentosum Group C Protein Complex Is the Initiator of Global Genome Nucleotide Excision Repair 
Kaoru Sugasawa, Jessica M.Y Ng, Chikahide Masutani, Shigenori Iwai, Peter J van der Spek, André P.M Eker, Fumio Hanaoka, Dirk Bootsma, Jan H.J Hoeijmakers 
Molecular Cell 
Volume 2, Issue 2, Pages (August 1998)
DOI: /S (00)80132-X

2. Figure 1
A Schematic Representation of the Damage Recognition-Competition Assay
(A) The earliest events of the normal NER process are depicted in a simplified hypothetical model.
(B) In vitro damage recognition-competition assay using two damaged DNAs. See text for details.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

3. Figure 2
Specificity of Fractionated XP Cell Extracts Utilized in the Present Studies
(A) A scheme for phosphocellulose fractionation of whole cell extracts.
(B) The purified human XPC-HR23B protein complex and the mouse XPA protein were subjected to SDS–polyacrylamide gel electrophoresis. Proteins were visualized by silver staining (XPC-HR23B) or by Coomassie Brilliant Blue staining (XPA).
(C and D) A mixture of AAF-damaged and nondamaged DNAs was incubated at 30°C for 60 min with CFII from XP cells and purified proteins as indicated, under the standard conditions for in vitro NER reaction. All reactions contained 100 μg of CFII fractions in total. Amounts of purified proteins used were 45 ng for XPC-HR23B, 20 ng for XPA, and 125 ng for RPA. DNA was purified from each reaction, and then the repair gaps were filled by T4 DNA polymerase in the presence of [α-32P]dNTP. The DNA samples were linearized and subjected to agarose gel electrophoresis followed by autoradiography.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

4. Figure 3
Competitive NER Assay Using CFIIs from XP-A and XP-C Mutant Cells
(A) Two AAF-damaged DNAs (DD-I and DD-II) were separately preincubated at 30°C for 15 min with the indicated protein fractions. After the two reactions were combined, nondamaged DNA (ND) and indicated protein fractions were added. Amounts of protein fractions used were 400 μg for each CFII and 250 ng for RPA. Aliquots of the mixtures were further incubated at 30°C for the indicated periods of time. Repair gaps generated during the second incubations were filled with T4 DNA polymerase and [α-32P]dNTP. The DNA samples were linearized and fractionated by agarose gel electrophoresis. A photograph of the ethidium bromide–stained gel and the corresponding autoradiogram are shown.
(B) DNA repair synthesis in each damaged DNA was quantitated. Radioactivity incorporated into ND was subtracted as a background from that in damaged DNA bands at each time point. The resulting values were further subtracted by those at the time point of 0 min and plotted as a time course. Closed circles, DNA repair synthesis in DD-I; open circles, DD-II.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

5. Figure 4 Competition between XPC-HR23B and XPA
(A, C, and E) Two AAF-damaged DNAs (DD-I and DD-II) or, for lanes 9–12 in (A), nondamaged version of pBS.XPCΔ (designated ND′) were separately preincubated at 30°C for 15 min with indicated protein fractions. The two reactions were combined, supplemented with indicated protein fractions as well as nondamaged pHM14 control (ND), and further incubated for various periods of time as indicated. Amounts of protein fractions used were 400 μg for each CFII, 90 ng for XPC-HR23B, 40 ng for XPA, and 250 ng for RPA. Repair gaps created in each damaged DNAs were labeled by T4 DNA polymerase. DNA samples were linearized and subjected to agarose gel electrophoresis followed by autoradiography.
(B, D, and F) Radioactivity incorporated in each band in (A), (C), and (E) was quantitated. DNA repair synthesis occurring in each damaged DNA was calculated as done for Figure 3B and depicted as graphs. Closed symbols represent repair of DD-I, while open symbols represent repair of DD-II. Closed circles, open circles: lanes 1–4; closed triangles, open triangles: lanes 5–8; closed squares, open squares: lanes 9–12.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

6. Figure 5 XPC-HR23B Preferentially Binds to Damaged DNAs
(A) A mixture of pBS.XPCΔ (nondamaged or damaged with various agents as indicated) and nondamaged pHM14 (200 ng each) was incubated in the presence (+) or absence (−) of 20 ng of XPC-HR23B. Affinity-purified anti-XPC polyclonal antibodies (0.5 μg) were added and precipitated with protein A–Sepharose beads. DNAs in the supernatant (unbound) and precipitate (bound) fractions were purified, linearized, and fractionated by agarose gel electrophoresis. The DNAs were transferred onto nylon membrane and visualized by hybridization to 32P-labeled pBKS sequence.
(B) The percentages of DNA recovered in the bound fractions were quantitated for lanes 2, 4, 6, and 8 in (A). The mean values and standard errors were calculated from two independent experiments. Solid bars, pBS.XPCΔ; shaded bars, pHM14.
(C) A mixture of 200 ng of nondamaged pHM14 and varying amounts of AAF-damaged pBS.XPCΔ was incubated with 20 ng of XPC-HR23B, and then immunoprecipitated with anti-XPC antibodies. Linearized DNAs in unbound and bound fractions were subjected to agarose gel electrophoresis followed by Southern blot analysis as done in (A).
(D) Quantitation of the result in (C). The mean values and standard errors were calculated from two independent experiments. (closed circles), AAF-damaged DNA; (closed squares), nondamaged DNA.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

7. Figure 6 Footprint Analysis of XPC-HR23B Bound to a UV-Induced 6-4PP
(A) Defined DNA substrate containing a single 6-4PP. Sequences of the oligonucleotides used for substrate preparation for DNase I footprinting assays are shown by boldfaced letters.
(B and C) Protection of 6-4PPs against DNase I digestion by XPC-HR23B. DNA fragments containing a site-specific 6-4PP were 3′-labeled at the Acc65I site for the damaged (top) strand (B) or at the BstXI site for the nondamaged (bottom) strand (C). The labeled DNA fragments were incubated with or without increasing amounts of XPC-HR23B, and then briefly digested by DNase I (or mock-digested). The amounts of XPC-HR23B used were 5 ng (lanes 3 and 8), 10 ng (lanes 4 and 9), and 20 ng (lanes 5 and 10). As markers, the “G” Maxam-Gilbert sequence ladders were prepared from the same labeled DNA fragments and electrophoresed alongside. The 6-4PP itself is sensitive to piperidine cleavage and gave heavy bands in the Maxam-Gilbert ladders (indicated by asterisks in [B]). In all panels, regions protected and cleavage enhanced by XPC-HR23B binding are indicated by solid lines and arrows, respectively. Arrowheads denote sites accessible to DNase I within the protected regions.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)

8. Figure 7 A Two-Stage Damage Recognition Model for NER
(i) A lesion located in the global genome (GGR) or in the transcribed strand of an active gene (TCR).
(ii) The lesion is first recognized by the XPC-HR23B complex (GGR) or RNA polymerase (TCR). XPC-HR23B may induce some conformational changes of the DNA helix in the vicinity of the lesion, which would favor the subsequent assembly of other NER factors.
(iii) XPA (possibly together with RPA) participates in the DNA–protein complex to verify the substrate specificity of the lesion. An involvement of TFIIH and initial opening of double-stranded DNA may precede the function of XPA (Evans et al. 1997). It is unknown whether XPC-HR23B is displaced from the lesion or remains bound to it. In TCR, CSA and CSB might be involved in this step.
(iv) The two DNA helicase subunits of TFIIH may fully open the double-stranded DNA around the lesion.
(v) Two structure-specific endonucleases, ERCC1-XPF and XPG, make dual incisions at the 5′ and 3′ sites, respectively.
Molecular Cell 1998 2, DOI: ( /S (00)80132-X)